Journal of Insect Physiology

The convergent lady beetle Hippodamia convergens is widespread in the Americas and considered an important beneficial insect due to its use in pest control. Early work on this species characterized the beetles as freeze-avoidant (freeze-intolerant), suggesting they survive low winter temperatures by physiologically preventing ice formation to temperatures as low as -15{degrees}C. Here, we show that H. convergens can be freeze-tolerant if ice formation occurs at relatively high temperatures. There was 100% survival following inoculative freezing at -0.5{degrees}C and exposure to -3{degrees}C for 20 hours, as well as freezing that spontaneously occurred in fed beetles exposed to -4{degrees}C for 4 hours. Males exposed to 0{degrees}C or -4{degrees}C for 4 hours had similar mating behaviours (latency to first mating, copulation duration) as control beetles exposed to 4{degrees}C, although sample sizes were too small to determine whether freezing itself had an effect on these behaviours. Several putative cryoprotectants were detected in fat body tissue of H. convergens: glycerol, proline, trehalose, and myo-inositol - in order of abundance. Exposure to -3{degrees}C for 20 hours, whether frozen or unfrozen, did not statistically affect cryoprotectant accumulation, although there was a trend towards increased glycerol following freezing. This study is the first to describe inoculative freeze tolerance in a lady beetle and establishes a baseline for future studies that examine the mechanisms underlying this freeze tolerance and the effects of freezing on reproductive behaviour. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/744689v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1c0382org.highwire.dtl.DTLVardef@12a5932org.highwire.dtl.DTLVardef@145f4adorg.highwire.dtl.DTLVardef@1c2cb22_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIHippodamia convergens can tolerate freezing at high subzero temperatures C_LIO_LIMating behaviour of males is normal after chilling or freezing C_LIO_LICryoprotectant accumulation may support overwintering survival C_LI

Winter poses harsh physiological challenges for insects living in temperate ponds due to the combination of low temperatures and reduced oxygen levels (hypoxia). Despite these stressors, water boatmen (Hesperocorixa sp.), backswimmers (Notonecta sp.) and diving beetles (Laccophilus maculosus) remain active year-round in eastern Nova Scotia, including in ice- covered ponds. However, the underlying mechanisms that allow pond insects to survive overwintering have not been widely studied. We hypothesized that cold and hypoxia tolerance would improve from September to March in these insects, with changes in whole-animal and biochemical correlates of tolerance to both stressors. We collected insects from the field and stocked them in outdoor freshwater mesocosms. Every two months, between September 2023 and April 2024, we characterized whole animal responses and biochemical changes. Whole insect cold tolerance assays indicated a trend of improved ability to sustain voluntary muscle control (CTmin) at lower temperatures in winter-collected insects and higher internal fluid freezing temperatures (SCP). There were no changes in whole animal correlates of hypoxia tolerance over time (surface respiration frequency, submersion and surface time). Potential cryoprotectants like proline, myo-inositol and trehalose increased in concentration in multiple insect taxa during winter but were relatively low compared to terrestrial insect studies. As lactate dehydrogenase activity did not change, there is little evidence that the insects experienced functional hypoxia sufficient to induce anaerobic metabolism. Overall, this research has established a baseline cold and hypoxia tolerance dataset in understudied pond insects. Summary StatementPond insects remain active during low temperature and low oxygen conditions associated with ice cover in winter, supported by physiological and biochemical changes.

Insects can experience a loss of fertility under acute heat stress, but impacts vary among species and depend on life stage and sex. We examined the effects of sublethal heat shocks on the fertility of three mosquito species (Aedes aegypti, Aedes notoscriptus and Culex quinquefasciatus) when males or females were exposed at the pupal and adult stages. Reproductive performance was assessed through egg-laying success, fecundity, egg hatchability and viable offspring production. Female heat shock had limited effects on fertility in all three species, though exposure of adult females to 41 {degrees}C for 1 hr reduced viable offspring production in Ae. notoscriptus. Male heat shock reduced egg hatchability in Cx. quinquefasciatus and viable offspring production in Ae. aegypti depending on the life stage exposed. We investigated several other aspects of heat shock exposure on Ae. aegypti fertility. In this species, male fertility partially recovered within 24-48 hours following heat shock exposure, while effects of heat shocks on female fertility occurred regardless of whether heat exposure occurred pre- or post-mating. In addition, maternal heat shock exposure enhanced offspring fecundity and viable offspring production under subsequent sublethal heat stress. Overall, our findings demonstrate that mosquito fertility is largely robust to short-term heat shocks, but there are also complex trait, sex, stage, temporal and species-specific effects, with implications for predicting mosquito population persistence and species persistence under future climate extremes.

BACKGROUND: Aphids damage crops through sustained feeding and by rapid transmission of plant viruses, processes that are not effectively prevented by conventional insecticides. The sugarcane aphid (Melanaphis sacchari), an invasive pest of sorghum in the United States, can inoculate non-persistent viruses within minutes of probing. Disrupting feeding phases represents an alternative strategy in plant disease and integrated pest management. ATP-binding cassette transporters (ABC transporters) influence insect physiological performance, but their role in regulating aphid feeding behavior relevant to crop damage and virus transmission remains unknown. This study evaluated whether ABC transporter activity alters feeding phases of M. sacchari on sorghum. RESULTS: Aphids exposed to ABC transporter modulators via artificial diet and monitored using Electrical Penetration Graph (EPG) recordings showed significant reductions in salivation in E1 phase, compared with controls (P < 0.05) and an increased duration in xylem phase when treated with elacridar, an ABC transporter inhibitor. Aphids treated with dexamethasone, an ABC transporter activator, exhibited increased time spent in pathway phase compared to the controls. ATPase assays also showed significant variations in modulator-treated samples compared to the controls. CONCLUSION: These results demonstrate that transporter modulators alter feeding behaviors which might lead to reduced crop damage/virus transmission, including pathway activities, salivation, and xylem ingestion. This study opens the path to identify ABC transporters as regulators of feeding phases, and to advance the understanding of aphid-plant interactions. This new knowledge will support the development of feeding-based pest management strategies to reduce crop damage and virus transmission.

Background: The insect cuticle is a chitin-containing extracellular matrix whose formation requires precise coordination of chitin biosynthesis, fibril organization, and post-synthetic maturation. Although lipids are essential components of the cuticle, the molecular mechanisms linking lipid transport to chitin deposition remain poorly understood. Here we identify TcFabp8, a fatty acid-binding protein in Tribolium castaneum, as a functionally important regulator of chitin synthase 1 (Chs1)-dependent cuticle formation. Results: RNAi-mediated depletion of TcFABP8 caused molting defects, reduced cuticle thickness, loss of apical TcChs1 immunoreactivity, and reduced chitin deposition and organization. The finding that TcCHS1 transcript levels were unaffected by RNAi targeting TcFABP8 suggests that a post-transcriptional step, potentially involving vesicular TcChs1 transport, is affected, thereby impairing chitin synthesis and deposition. Synchrotron X-ray diffraction analysis of adult elytra revealed that TcFabp8 depletion preserved characteristic -chitin-associated scattering features, but altered chitin fibril accumulation, crystalline coherence, and the spatially ordered alignment of fibrils within the cuticular matrix. The TcFabp8 gene locus encodes splice variants yielding different N-terminal architectures: TcFabp8X1 carries a Sec/SPI signal peptide which is absent in TcFabp8X2 and TcFabp8X3. Immunocytochemistry using TcFabp8-specific antibodies detected intracellular signals in subapical cytoplasmic puncta and extracellular signals within the cuticle layers, which partially colocalized with Nile red fluorescence indicating lipid-rich environments. Conclusions: Together, our data demonstrate that TcFabp8 is a critical facilitator of TcChs1-dependent chitin deposition and extends functional connection between lipid transport and chitin biosynthesis in insects.

Smoke flies (Diptera: Platypezidae: Microsania) are among the most specialized pyrophilic insects, exhibiting a strong and rapid reaction to smoke. Although their attraction to smoke has been known for a century worldwide, their immature stages and breeding habitat remained undiscovered. We hand-netted smoke flies at seven prescribed forest burns in boreal Finland between 2022 and 2024 and took soil samples in 2026. Smoke fly swarming and reproductive behaviour was observed at all burn sites. We obtained smoke flies of five out of the six Finnish species, with Microsania pallipes dominating (94%). Following the behavioural observations at burn sites, we later screened soil samples and found larvae identified as Microsania mtDNA COI barcodes in the burnt soil. We provide a first description of the larva of Microsania straeleni (Microsaniinae). Our discovery of the larval habitat in burnt soil resolved the long-standing knowledge gap on smoke fly breeding sites. The morphological characteristics of the immature stages will help elucidate the phylogenetic placement of the Microsaniinae within the Platypezoidea lineage.

The generalist Helicoverpa armigera and the specialist Helicoverpa assulta are closely related noctuid pests and are among the few insect herbivores capable of feeding on and damaging hot pepper fruits, which contain the defensive compound capsaicin. Cytochrome P450 monooxygenases (P450s) contribute to the metabolism of plant defensive compounds and can facilitate insect adaptation to chemically defended host plants. Here, we identified CYP6AN1 in H. assulta (HassCYP6AN1) and comparatively characterized the CYP6AN1 orthologs from H. armigera (HarmCYP6AN1) and H. assulta. RACE identified one full-length HarmCYP6AN1 transcript and three full-length HassCYP6AN1 transcript isoforms. The HassCYP6AN1 isoforms contained distinct 5' UTRs generated by alternative transcription initiation and splicing but shared an identical coding sequence. Sequence comparisons and phylogenetic analysis supported their assignment as orthologs. Constitutive CYP6AN1 expression was higher in the H. assulta midgut, whereas dietary capsaicin significantly induced CYP6AN1 expression in the H. armigera midgut. Recombinant CYP6AN1 proteins were co-expressed with NADPH-cytochrome P450 reductase in Escherichia coli, and their substrate-metabolizing activities were evaluated using HPLC-based depletion assays. Both orthologs metabolized capsaicin, but HarmCYP6AN1 exhibited an approximately 2.3-fold higher depletion activity than HassCYP6AN1 under the conditions tested. HarmCYP6AN1 also showed P450-content-dependent xanthotoxin depletion, whereas no detectable xanthotoxin metabolism was observed for HassCYP6AN1. These findings establish CYP6AN1 as a component of the capsaicin-metabolizing repertoire of both species and reveal substantial divergence between the orthologs in transcript organization, expression regulation, catalytic activity, and detectable substrate range.

Odors allow animals to evaluate resources and risks, but the neural and inherited consequences of experience with ecologically meaningful odors remain poorly understood. Since both parasitoids and entomopathogenic fungi are widely used as agents in integrated biocontrol, we asked whether exposure to pathogen-associated volatiles can reassign odor value in a parasitoid wasp and whether the acquired bias is transmitted to descendants. Adult female Scleroderma guani were exposed, without host contact, to volatiles from Beauveria bassiana-infected hosts or to methyl cinnamate (MC), a representative component of the infected-host odor. Twelve-hour exposure shifted subsequent choices away from infected-host odor and MC, whereas shorter exposure produced different effects, indicating duration-dependent olfactory plasticity. The shift occurred without an explicit reward or punishment and is therefore consistent with non-associative learning. Unexposed F1 and F2 descendants of MC-exposed females expressed the same avoidance bias. MC stimulation elicited stronger calcium responses in mushroom-body regions of trained females and their descendants. Head transcriptomes identified reduced expression of the crustacean cardioactive peptide receptor (CCAPR) as a shared molecular signature, and CCAPR RNA interference phenocopied the odor-avoidance behavior. Finally, maternal treatment with sinefungin, a broad S-adenosylmethionine-dependent methyltransferase inhibitor, left maternal behavioral shift intact but abolished the F1 bias, implicating a methylation-sensitive process in transmission. Together, these results reveal that a pathogen-associated odor can induce durable, cross-generational reweighting of olfactory value in a non-model insect and identify candidate neural and neuropeptidergic mechanisms linking sensory experience to inherited behavior, and providing a new perspective on utilizing training in integrated biocontrol as well.

Over the course of evolution, many species have adapted to the environmental conditions found at higher latitudes. This is likely also true of the circadian system, which regulates daily behavior and helps measure day length to facilitate adaptation to the seasons, though how the circadian clock adapts to latitude remains largely unknown. Here, we investigated daily activity patterns under long photoperiods and in constant darkness, as well as the underlying circadian clock network, in 52 Drosophila species from around the world. We found a strong, significant positive correlation between the clock's plasticity, measured as the phase difference between morning and evening activity, and the average geographical latitude from which the species originate. The mechanisms underpinning this increase in plasticity differed between species: in the subgenus Siphlodora, the clock weakened with increasing latitude, accompanied by a reduction of the neuropeptide Pigment-Dispersing Factor in the small ventrolateral clock neurons (s-LNv), whereas in the subgenus Drosophila the opposite was true. The subgenus Sophophora, by contrast, showed a negative correlation between circadian plasticity and PDF levels in the s-LNv axons. These results suggest that Drosophila species have evolved diverse, independent strategies to adapt their circadian clock to higher latitudes.

The physiology of ectotherms can change substantially during acclimation to changing environmental temperature. The role of transcription in acclimation responses has been well-established, but it remains unclear whether transcriptional regulation generally reflects abrupt changes after surpassing temperature thresholds, or whether transcript abundance is a relatively monotonic, continuous function of acclimation temperature. In this study we exposed adult male Drosophila subobscura flies to four different 96-hour acclimation treatments at temperatures that were not acutely stressful but ranged from relatively cold (10{degrees}C) to relatively warm (27{degrees}C) with respect to standard rearing conditions. Transcriptome sequencing of whole-body homogenates (mRNAseq) revealed a massive, transcriptome-wide response across acclimation temperatures, with a marked overrepresentation of genes that were continuously and monotonically up- and down-regulated in response to increasing acclimation temperature. Though some genes showed more complex relationships consistent with putative threshold responses, a high percentage of the differentially expressed transcriptome (42%) showed continuous and strictly monotonic relationships. Functional enrichment suggested continuous up-regulation of spermatogenesis-related transcripts with increasing temperature and continuous up-regulation of oxidative phosphorylation-related transcripts with decreasing temperature, illustrating contrasting patterns consistent with previous studies of thermal sensitivity of male reproduction and metabolic compensation in the cold. Thus, continuous thermal sensitivity of transcription is a hallmark of acclimation in D. subobscura that likely underlies the continuous thermal sensitivity of downstream physiological processes. We also provide evidence for shared transcriptomic responses across short-term acclimation (this study) vs. published results for long-term, developmental acclimation.

Corazonin is implicated in regulating diverse behavioral and physiological responses in insects, yet its role in honey bees remains unclear. Work in social and non-social species suggests an ancestral function for corazonin as a metabolic stress signal and a derived function as a pro-foraging signal in social insects. To investigate this hypothesis, we examined corazonin signaling across worker roles and metabolic states and tested its regulatory relationships with pathways central to worker division of labor. Central corazonin levels (both transcript and peptide) were approximately threefold higher in foragers than in nurses. Acute metabolic challenge did not alter brain corazonin but strongly upregulated fat body corazonin receptor (CrzR) transcript in nurses only, suggesting role-specific induction of peripheral sensitivity to corazonin signaling. Injection of synthetic corazonin decreased fat body insulin receptor substrate transcript, while transcripts of vitellogenin (Vg), unpaired-like A, ecdysone receptor B, and Cyp314a1 were unaffected. Measures of gustatory responsiveness, starvation resistance, and hemolymph sugar levels were similarly unaffected by corazonin injection. Reducing Vg through RNA interference in nurse-age bees did not affect CrzR transcript levels, despite vitellogenin's established roles in stress buffering, antioxidant defense, and lifespan. This contrast highlights the specificity of CrzR upregulation in nurses as a response to metabolic challenge rather than general stress. Together, these findings support corazonin as a co-opted nutritional signal that helps shape the forager physiological state, suggesting that it may act in part through interactions with insulin signaling rather than directly regulating classical nutritional or stress-response pathways.

The red palm weevil, Rhynchophorus ferrugineus, is the most economically destructive palm pest worldwide, threatening livelihoods, food security, and ecosystems across 49 countries. Weevil management currently relies predominantly on chemical insecticides, raising significant environmental and public health concerns. Despite its global agricultural importance, genetic approaches to pest management and the mechanistic basis of genome-editing strategies in Rhynchophorus remain largely unexplored. Here, we employed CRISPR/Cas9 genome editing to disrupt the R. ferrugineus ommochrome biosynthetic pathway -- a multi-enzymatic metabolic cascade that converts tryptophan into ommochrome pigments, including brown, yellow, and red pigments. We targeted two key pathway components: the ATP-binding cassette transporter white and the heme peroxidase cardinal. Both genes were ubiquitously expressed, with peak expression levels in the gut, fat body, and head. Elevated transcript levels were observed across early, mid, and late pupal stages and in 0-, 1-, and 2-day-old adult males and females, consistent with the progression of eye pigmentation throughout the R. ferrugineus life cycle. Embryonic microinjection of a single guide RNA (sgRNA)-Cas9 ribonucleoprotein complex targeting white produced in the Generation-0 (G0) adults with a distinct, white-eyed phenotype with a brownish outer margin, in contrast to the black eyes of wild-type adults. Genome-edited cardinal mutant adults displayed a translucent, brownish-white-eyed phenotype, with white streaks that gradually transitioned to a persistent translucent reddish-brown eye coloration. Mutations in both genes were confirmed in G0 adults by genomic DNA sequencing. Mutant adults were crossed to generate heterozygous G1 (+/-), G2 (-/-, -/+, and +/+), and G3 lines (-/-) with genotypes verified as carrying 2-, 3-, 9-, and 13-nucleotide deletions. A stable, heritable eye-color phenotype was established in homozygous knockout (-/-) G3 lines for both white and cardinal, confirmed by unambiguous indel (insertions/deletions) genotyping. Inheritance analysis revealed that both genes are X-linked, following a classical Mendelian sex-linked pattern: paternal alleles are transmitted exclusively to daughters, while maternal alleles are inherited equally by both daughters and sons. This study establishes the first fully homozygous knockout strain in R. ferrugineus and, by characterizing sex-linked inheritance in a coleopteran system, advances our understanding of how CRISPR/Cas9 can be efficiently applied to destructive palm weevil species. The present study represents the first report of CRISPR/Cas9 genome editing in any weevil (Curculionidae), using white and cardinal as marker genes. These findings provide a valuable platform for functional genomics and genome engineering in R. ferrugineus and offer a translational framework for genome editing in the invasive South American palm weevil, R. palmarum, laying a solid foundation for the development of gene-drive strategies aimed at sustainable palm weevil population control.

The global expansion of Black Soldier Fly (BSF), Hermetia illucens (L.), production systems for organic waste management has increased the need to better understand its reproductive biology in order to optimize mass-rearing programs. In this study, we macroscopically characterized chronological morphological changes in the internal genitalia of adult H. illucens reared on a sustainable alternative larval diet based on coffee pulp and corn bran. Morphological assessments and dissections were conducted on female and male reproductive tracts at 3, 6, and 9 days after adult emergence. Overall, the general organization of both reproductive systems was consistent with previous descriptions. A notable observation was the presence of a tripartite fertilization chamber in females, composed of three distinct compartments apparently associated with the three spermathecae. The functional significance of this anatomical organization remains to be determined. Females showed progressively advanced ovarian development and reached the clearest morphological indicators of reproductive maturity at 9 days, while males showed the greatest testicular distention and opacity at the same age. Compared with maturation times reported in previous studies using conventional larval diets, these observations suggest a later pattern of reproductive maturation under the coffee pulp-based diet. The observed differences may be associated with the nutritional composition and carbohydrate-to-protein balance of the larval diet. These results provide chronological and iconographic information that may contribute to the optimization of laboratory rearing protocols and the use of coffee by-products in H. illucens bioconversion systems.

Foraging hematophagous flies such as the stable fly, Stomoxys calcitrans, are shown or expected to sense multiple characteristics of visual host cues. These include relative intensity, spectral composition, and the angle and degree of polarization. Here, we investigated the role of each of these visual cue characteristics on stable fly attraction. In laboratory two-choice bioassays, we offered groups of female or male stable flies a choice between paired photostimuli that differed in their visual characteristics, recording the flies' landing responses on stimuli as the response criterion. Bioassay flies preferentially selected lower-intensity visual stimuli even when paired stimuli markedly differed in spectral composition and polarization characteristics (a high degree of horizontal or vertical polarization). When paired stimuli had identical relative intensity, flies failed to discriminate between the stimulus that presented ultraviolet, blue, green, and red wavebands and a corresponding paired stimulus lacking one of these wavebands. Our results support the hypothesis that foraging stable flies may respond to colour and polarization characteristics of visual stimuli as modulations of perceived intensity. Acquired knowledge on the mechanisms that underlie stable fly visual attraction may inform the design of vision-based traps so that they become optimally attractive to stable flies and other biting flies.

Many social insects, such as ants, change their behavior stereotypically from nursing to foraging as they age, a concept known as temporal polyethism. Biogenic amines are associated with these specific behaviors, but much about them remains poorly understood in these animals. Many aminergic systems lack anatomical characterization, and little is known about the expression of genes required for the aforementioned behavioral phenotypes. Here, we studied Pogonomyrmex barbatus brains and identified the neurons that produce two relevant amines, dopamine and octopamine, by detecting their synthesis enzymes, tyrosine hydroxylase and tyramine beta-hydroxylase, and their transcripts. We also compared the expression of both genes in young nurses and mature foragers by measuring the fluorescence intensity. Dopaminergic and octopaminergic neurons are predominantly located in the protocerebrum and in the subesophageal zone. Neurons that produce dopamine are also present in the optic lobes, whereas octopamine-producing ones show clusters in the antennal lobes. Both genes are downregulated as the organism ages. A reduction in the expression of these genes might be correlated with the mature workers increased propensity to perform extranidal tasks, such as foraging.

Planning husbandry tasks and experiments with Drosophila melanogaster requires converting a target date into development times that depend on the rearing temperature. This calculation needs to be done for each cross, genotype, and temperature, and the risk of error grows quickly. Available laboratory management tools let users register stocks, crosses, and track them, but they do not create schedules based on a clear, adjustable thermal model. To fill that gap, we developed DrosoTracker, a self-contained web application that works offline and predicts Drosophila development with a thermal summation model recalibrated through regression on data from Powsner (1935) (T0 = 11.78 {degrees}C, DD = 116.38 {degrees}C{middle dot}days, R{superscript 2} = 0.997). The model offers an optional two-level calibration driven by user observations. A wild-type strain first adjusts the model to the laboratorys own conditions. Then each genotype is calibrated against that reference using a random-effects shrinkage estimator that accounts for measurement error and between-batch variability. The model creates schedules for husbandry tasks, evaluates adult cohort survival with the Kaplan-Meier estimator and the log-rank test, and calculates sample size for lifespan studies using Schoenfelds formula. The quantitative components were checked against independent references, including Rs survival package and manual calculations. Ongoing work is focused on validating the calibrated model using cohorts specifically bred for this purpose. DrosoTracker runs entirely in the browser, stores data locally, and is available in English and Spanish.

Nesidiocoris tenuis is an important zoophytophagous mirid bug used as a biological control agent in agriculture, and breeding efforts based on genomic information aim to increase its utility. Visible eye-color mutants are useful genetic markers because they are easily distinguishable and are therefore widely used in insect genetics and genome editing studies. Here, we investigated the genetic basis of a spontaneous red-eye mutant identified in a laboratory strain of N. tenuis. Classical crossing experiments suggested that the red-eye phenotype is controlled by a single recessive locus. RNA-seq and RNA interference (RNAi) analyses identified scarlet and cinnabar as the primary candidate genes associated with the phenotype. Further genomic analysis revealed a large deletion and insertion within exon 5 of the mutant scarlet allele, potentially causing exon skipping and disrupting transporter structure. The insertion pattern is consistent with a microhomology-mediated break-induced replication (MMBIR)/fork stalling and template switching (FoSTeS)-like event that may have been generated through polymerase{theta} -mediated repair. Together, these findings identify the causative mutation underlying the red-eye phenotype and provide a useful visible marker for future functional genetic studies and genome-assisted breeding in N. tenuis.

Ethanol is naturally present in nectar, but we still know little about how pollinators cope with long-term exposure to it at low concentrations. We exposed adult worker honeybees (Apis mellifera carnica Poll.) for eight days to diets containing 0, 0.1, 0.5, 1, 2, or 10% ethanol and measured survival, estimated intake, open-field locomotion, sting extension, whole-body trehalose, and hemolymph ethanol. Hemolymph ethanol was correlated with dietary concentration, attesting to internal exposure. Survival differed among diets, an effect which was driven by the difference between the 10% ethanol treatment and the control. Food intake was lowest at the 10% ethanol diet. There was also a modest increase in intake under the 0.1% ethanol treatment. In terms of locomotor features, bees moved less, moved more slowly, explored less space, covered shorter distances, and followed more tortuous paths after the 10% ethanol treatment. Neither sting extension nor trehalose were affected by dietary ethanol. The results showed no beneficial stimulation at lower concentrations. Instead, performance was endpoint-specific: at 0.1-2% ethanol in the diet, performance remained relatively stable across most parameters, whilst clear impairment was evident at 10%. These findings highlight resilience to repeated ethanol exposure in honeybees.

Mosquitoes use their sensitive hearing in a well-described courtship and mating behavior, but their ability to discriminate other stimuli relevant to their survival and reproduction via this sensory channel remains underexplored. Here, we characterize a rapid climb response in free-flying mosquitoes elicited by loud low-frequency sounds (tested at 180Hz and 300Hz) but not by tones of equivalent acoustic loudness reflecting conspecific female (450Hz) and male (700Hz) wingbeat frequencies. Presentation of the same sounds in tethered flight elicited modulation of both wingstroke amplitude and frequency components similar in magnitude to those observed in response to a looming visual stimulus. These tones are spectrally consistent with recorded audio of a dragonfly takeoff, playback of which is sufficient to release the behavior. Taken together, these data are consistent with the hypothesis that mosquitoes use their hearing to detect the acoustic signature of aerial predators such as dragonflies.

Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) is one of the most dangerous palm pests worldwide. Here we report the invasion process of R. ferrugineus in Uruguay. Beyond morphological and molecular identification, we infer the geographic origin of the invasive population, describe the succession of introduced and native palms attacked, provide an easy-to-use key to differentiate the invader R. ferrugineus from the native Rhynchophorus palmarum, and model the ecological niche of R. ferrugineus in Uruguay and neighboring countries. First detected in 2022, the Uruguayan population of the weevil is the first to establish on mainland America, following the earlier island records from Curacao and Aruba. Phylogenetic analysis showed that the Uruguayan R. ferrugineus sequences clustered within a single clade, most closely related to sequences from southern China, and clearly distinct from the sequences of Curacao and Aruba, indicating at least two independent introduction events into the Americas from different source regions. After four years, R. ferrugineus has spread across an area of nearly 80,000 square kilometers and in January 2026 it reached Argentina, invading two points near the border with Uruguay. Over the first two years after the invasion, attacks focused on Phoenix canariensis, yet the three most common native palms in Uruguay, Butia odorata, Butia yatay, and Syagrus romanzoffiana, are currently being attacked. The diagnostic key to differentiate the pair R. palmarum / R. ferrugineus is based on three characters: the tip of the rostrum, the tegminal plate, and the ratio between the interocular space and the width of the rostrum at the base. Niche models showed that Uruguay and the neighboring areas of Argentina and Brazil are suitable for R. ferrugineus, and these are precisely the areas where both the highly susceptible P. canariensis and the native B. odorata, B. yatay, and S. romanzoffiana are present. Neighboring countries should therefore be prepared for the expansion of R. ferrugineus in the short term.